The recent perfection of the ability to incorporate pieces of DNA from any source into bacterial episomes and replicate them extensively is a major new advance of biotechnology with significant implications for both basic and applied research. This project will apply this new procedure to an analysis of sea urchin gene structure and function. We will exploit the new procedure to answer basic questions concerning the mechanisms of gene regulation and to help lay the foundation necessary to develop the complex technology which must be worked out to use the procedure for "genetic engineering" to deal directly with such medical problems as genetic deficiencies or cancer. We will incorporate random pieces of sea urchin DNA into bacterial episomes and, using DNA-RNA hybridization of messenger RNA and heterogeneous nuclear RNA from various stages of development, select episomes that carry genes which are transcribed at one or all of the various stages of development. The ratio of unique and repetitious DNA in each episome will be determined by total sea urchin DNA excess reannealing of radioactive episomal DNA. The percentage of each episome which is transcribed into mRNA and hnRNA at each stage of development will be determined by saturation hybridization. The arrangement of the transcribed sequences will be determined by electron microscopy of the DNA-RNA hybrids. These results should elucidate spatial relationships of hnRNA to mRNA sequences and of different mRNA sequences to each other. The episomes will be returned to the sea urchin cytoplasm using liposomes and their expression and regulation examined. Studies on the isolation of hemoglobin genes from rabbits and the nature of the complete gene for a specialized protein will also be carried out. These studies will contribute to basic research on the nature of the organization of the genome and on the mechanisms of gene regulation during developmemt. They also will lay the foundation for application of the techniques for cloning DNA to higher organisms so they may be used to deal with genetic engineering problems such as the addition of functional genes to the genome of individuals with defective genes or the addition of genes conferring a resistance to cancer viruses to the genomes of individuals.